中国组织工程研究

中国组织工程研究 ›› 2022, Vol. 26 ›› Issue (5): 756-761.doi: 10.12307/2022.123

• 组织构建细胞学实验 cytology experiments in tissue construction • 上一篇    下一篇

miR-373下调转化生长因子βⅡ型受体表达抑制肝星状细胞活化

徐  静1，严永敏2，蔡梦洁2，3   

  1. 1江苏大学附属医院检验科，江苏省镇江市   212013；2江苏大学医学院，江苏省镇江市   212013；3苏州大学附属第一医院，江苏省血液研究所，江苏省苏州市  215006
  • 收稿日期:2021-02-04 修回日期:2021-02-22 接受日期:2021-03-31 出版日期:2022-02-18 发布日期:2021-11-03
  • 通讯作者: 蔡梦洁，硕士，江苏大学医学院，江苏省镇江市 212013；苏州大学附属第一医院，江苏省血液研究所，江苏省苏州市 215006
  • 作者简介:徐静，女，1982年生，江苏省如皋市人，汉族，2008年江苏大学毕业，硕士，副主任技师，主要从事血清非编码RNA检测与消化道疾病诊断的研究。
  • 基金资助:
    国家自然科学基金面上项目(81670549)，项目负责人：严永敏

miR-373 inhibits hepatic stellate cell activation by downregulating transforming growth factor beta type II receptor 

Xu Jing1, Yan Yongmin2, Cai Mengjie2, 3    

  1. 1Department of Laboratory Medicine, Affiliated Hospital of Jiangsu University, Zhenjiang 212013, Jiangsu Province, China; 2Medical College of Jiangsu University, Zhenjiang 212013, Jiangsu Province, China; 3Jiangsu Institute of Hematology, First Affiliated Hospital of Soochow University, Suzhou 215006, Jiangsu Province, China
  • Received:2021-02-04 Revised:2021-02-22 Accepted:2021-03-31 Online:2022-02-18 Published:2021-11-03
  • Contact: Cai Mengjie, Master, Medical College of Jiangsu University, Zhenjiang 212013, Jiangsu Province, China; Jiangsu Institute of Hematology, First Affiliated Hospital of Soochow University, Suzhou 215006, Jiangsu Province, China
  • About author:Xu Jing, Master, Associate chief technician, Department of Laboratory Medicine, Affiliated Hospital of Jiangsu University, Zhenjiang 212013, Jiangsu Province, China
  • Supported by:
    the National Natural Science Foundation of China (General Program), No. 81670549 (to YYM)

PDF

87

摘要：

文题释义：
肝星状细胞：存在于肝窦间隙，正常情况下处于静止状态，当肝脏受到炎症或机械刺激等损伤时，肝星状细胞被激活，激活的肝星状细胞一方面大量增生并分泌胶原，另一方面通过细胞收缩使肝窦内压升高，最终奠定肝纤维化发展的病理基础。
转化生长因子βⅡ型受体：转化生长因子β是目前已知与纤维化、肿瘤关系极为密切而具有代表性的细胞因子，而转化生长因子β受体又在转化生长因子β信号传导、生物学效应等方面起重要作用，尤其是转化生长因子βⅡ型受体水平的变化影响转化生长因子β的功能。


背景：肝星状细胞活化分泌大量胶原是肝纤维化进展的主要因素，miRNA在肝星状细胞活化、凋亡等方面发挥着重要作用，miR-373调控肝星状细胞胶原分泌的作用及机制尚不清楚。
目的：探讨miR-373靶向调控转化生长因子βⅡ型受体在肝星状细胞胶原分泌中的作用。
方法：①建立Bal b/c小鼠肝纤维化模型，Masson法测定肝组织胶原，采集小鼠血清样本，提取血清总RNA，qRT-PCR定量分析小鼠    mmu-miR-291a-3p(人hsa-miR-373-3p的同源序列)水平。免疫组化检测肝组织转化生长因子βⅡ型受体和αSMA表达；②TargetScan软件预测miR-373的靶基因转化生长因子βⅡ型受体，采用双荧光素酶报告基因实验检测miR-373调控转化生长因子βⅡ型受体启动子的活性；③用25，50 nmol/L miR-373 mimics转染人肝星状细胞(LX-2)过表达miR-373，并以转染随机miRNA序列做对照，采用Western blot和免疫荧光检测转化生长因子βⅡ型受体和成纤维细胞激活蛋白的蛋白表达。实验方案经江苏大学实验动物伦理委员会批准(批准号为UJS-IACUC-AP-2020033127)。
结果与结论：①健康对照组相比，肝纤维化组小鼠血清mmu-miR-291a-3p水平明显降低(P < 0.001)，肝组织胶原合成显著增加；②肝纤维化组织转化生长因子βⅡ型受体和αSMA共表达显著增加；③miR-373显著抑制了转化生长因子βⅡ型受体启动子的活性(P < 0.01)，miR-373过表达显著抑制LX-2的转化生长因子βⅡ型受体(P < 0.05)和成纤维细胞激活蛋白(P < 0.01)的蛋白表达；④结果说明，miR-373通过调控转化生长因子βⅡ型受体表达抑制肝星状细胞活化和肝组织胶原沉积。

缩略语：转化生长因子βⅡ型受体：transforming growth factor-beta receptor type 2，TGFβR2

https://orcid.org/0000-0002-3438-2735 (徐静) 

关键词: 肝纤维化, miRNA, miR-373, 转化生长因子βⅡ型受体, TGFβR2

Abstract: BACKGROUND: Activated hepatic stellate cells can secrete a large amount of collagen, which is the main factor in the progression of liver fibrosis. miRNA plays an important role in the activation and apoptosis of hepatic stellate cells. The role and mechanism of miR-373 in regulating collagen secretion from hepatic stellate cell are still unclear.
OBJECTIVE: To investigate the role of miR-373 in the regulation of transforming growth factor beta type II receptor (TGFβR2) in collagen secretion from hepatic stellate cells.
METHODS: Bal b/c mouse model of liver fibrosis was established by CCl4 injection. Masson staining was used to detect the collagen content of liver tissues. Serum samples from liver fibrosis mice were collected and total RNA was extracted by Trizol method. Quantitative reverse transcription PCR (qRT-PCR) was used to analyze the expression level of mouse mmu-miR-291a-3p (the homologous sequence of human hsa-miR-373-3p). The expression of TGFβR2 and α-smooth muscle actin in liver tissue was detected by immunohistochemistry. TargetScan software was used to predict the target gene of miR-373. The dual luciferase reporter gene assay was used to detect the activity of miR-373 regulating TGFβR2 promoter. Human hepatic stellate cells (LX-2) were transfected with miR-373 mimics (25 and 50 nmol/L) to overexpress miR-373. LX-2 cells transfected with random miRNA sequence served as a control. Western blot and immunofluorescence were used to investigate the effect of miR-373 on the expression of TGFβR2 and fibroblast activation protein. The study protocol was approved by the Experimental Animal Ethics Committee of Jiangsu University (approval No. UJS-IACUC-AP-2020033127).
RESULTS AND CONCLUSION: Serum mmu-miR-291a-3plevel was significantly decreased in the fibrosis group compared with the health control group (P < 0.001), and collagen synthesis in the liver was significantly increased. Co-expression of TGFβR2 and α-smooth muscle actin in liver fibrosis tissue was obviously increased compared with the control group. miR-373 significantly inhibited the activity of TGFβR2 promoter in LX-2 cells (P < 0.01). The expression of TGFβR2 and fibroblast activation protein in LX-2 cells was significantly downregulated by miR-373 overexpression (P < 0.01). To conclude, miR-373 can inhibit the activation of hepatic stellate cells and collagen deposition in liver tissues by downregulating the expression of TGFβR2.

Key words: liver fibrosis, miRNA, miR-373, transforming growth factor beta type II receptor, TGFβR2

中图分类号: